The PTH/PTHrP receptor (PTHR), a G protein-coupled receptor (GPCR) of the B family, transmits both parathyroid hormone (PTH) and PTH-related Peptide (PTHrP) signals to initiate and regulate vital biochemical processes in bone and renal physiology. It is unknown how this single receptor discriminates between the two ligand signaling systems: PTH; endocrine and homeostatic, and PTHrP; a paracrine mediator of developmental and diverse organ biology. It is unclear also why in a clinical setting PTH(1-34) stimulates more prolonged increases in serum levels of 1,25-dihydroxy-vitamin-D, calcium, and bone resorption markers than does PTHrP(1-36), when the ligands are administered by continuous infusion so as to mimic conditions of primary hyperparathyroidism and humoral hypercalcemia of malignancy. We now advance a comprehensive model to account for these activities. Our recent studies show that PTH(1-34) differentiates itself from PTHrP(1-36) by inducing prolonged cAMP responses in cultured cells, and in vivo, which are mediated at the receptor level, and not by extended bioavailability of ligands. We discovered that during the time frame of cAMP production, PTHrP(1-36) action, is restricted to the cell surface, whereas PTH(1-34) trafficked to internalized sub-cellular compartments where it forms a stable complex with the PTHR, and continues to stimulate cAMP production. Such marked differences provide a mechanistic basis whereby PTH and PTHrP induce distinctly different responses and suggests that PTHR signaling to cAMP can continue from intracellular domains. Based on these novel findings, we propose the central hypothesis that cAMP production by the PTHR occurs both at the plasma membrane and from intracellular domains, with distinct lifetimes that have different consequences for cell signaling. This concept, supported by our recent findings and preliminary data described here, challenge the classical paradigm that cAMP production triggered by GPCRs originates exclusively at the cell membrane. The proposed experiments seek to determine a) the mechanisms of sustained cAMP responses triggered by the PTHR; and b) the consequences of sustained cAMP levels for cell signaling. These will be experimentally tested in the native environment of living cells by using optical technologies, as well as pharmacological, biochemical and proteomic approaches. These experiments will contribute to a fundamental understanding of the molecular and trafficking mechanisms of activation and signaling of the PTH/PTHrP/PTHR system in the native environment of living cells, which are needed to guide the development of safer and more specific and effective drugs for bone and mineral diseases. PUBLIC HEALTH RELEVANCE: The goal of this research plan is to understand how the medically important parathyroid hormone receptor transmits both parathyroid hormone (PTH) and PTH-related peptide (PTHrP) signals to regulate different physiological processes: PTH, endocrine and homeostatic regulates concentrations of calcium, phosphate ions, and vitamin D in blood and extracellular fluids, and PTHrP, a paracrine mediator of developing tissues such as bone. Pharmacological, biochemical and biophysical methods will be employed to advance a comprehensive model at the molecular and cellular levels to account for the functional differences between PTH and PTHrP, which will guide new therapies for osteoporosis.